Alzheimer's disease (AD) patients display a wide range of brain abnormalities. One of the hallmarks of AD is the florid deposition of senile plaques composed chiefly of Abeta. In some individuals familial early-onset AD (FAD) is attributable to specific mutations in the Amyloid Precursor Protein (APP) gene. Recently, it has been shown that Abeta levels are affected by mutations in the Presenilin 1 and 2 genes (PS1 and PS2). This argues strongly that AD may involve disturbances in APP metabolism. Correspondingly, there is tremendous interest in understanding the molecular actions of genes and agents that regulated Abeta production. Toward this goal much progress has been made. However, much remains to be elucidated regarding the subcellular mechanisms by which Abeta production is modulated, particularly in neurons (the cells most affected by AD). Post-translational processing of APP is highly complex, involving pathways that function in cell-type specific ways. One of the primary goals of this project is to dissection and analyze the subcellular pathways that process APP and Abeta in neurons harboring mutations in PS1. To accomplish this, APP processing will be examining in primary neurons derived from transgenic mice with PS1 M146V mutation "knocked into" the endogenous murine PS1 gene. These cells will be infected with vectors (Semliki Forest virus and Herpes Simplex Virus-1/amplicons) engineered to express wild-type and FAD-related mutations in APP and PS1, as well as APP trafficking/sorting mutations. Similarly, neurons from PS1-deficient mice will be examined to further assess the role of PS1 in APP processing. Interestingly estrogen, a substance with a number of neurobiological activities, has been shown to reduce Abeta production. To gain insight into the mechanisms by which estrogen opposes the generation of Abeta, another goal of this project is to identify the APP/Abeta processing pathways that are affected by the action of estrogen. These studies will be carried out in cultured primary rat neurons and the human neuronal NT2N cell line. These studies will likely provide new insights into the specific subcellular organelles and trafficking pathways that modulate neuronal APP and Abeta processing. This information may prove important in designing new strategies to prevent or control the pathological events underling Alzheimer's disease.